A European lab combines "light sheet" microscopy with an illumination process that subtracts the static caused by scattered photons to devise a way to clearly observe the inner workings of cells over a period of days

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Using a revolutionary new microscope, scientists can now peer into embryos and watch, in one of the world's smallest 3-D movies, as brains, eyes and other organs form. A team at the European Molecular Biology Laboratory (EMBL) in Heidelberg, Germany, watched zebra fish and fruit fly embryos develop under the scope for as long as 58 hours, charting the location of every cell as it danced around the embryo. This experiment would have been impossible a mere two years ago before a recent spate of innovations advanced microscopy years into the future.

When it comes to watching the inner workings of cells, fluorescence microscopy is second to none. In this technique, scientists attach fluorescent tags to cellular proteins and, by shining a laser on the cells, cause them to light up.

But placing cells under a standard fluorescent microscope essentially sentences them to death. Damaged by the scope's powerful laser, many perish before a few hours pass, so watching any extended process is difficult. In addition, cells under stress often behave differently than normal cells do, a huge stumbling block for scientists trying to draw connections between their experiments and the natural world.

The team at EMBL, headed by Ernst Stelzer, is part of a growing effort to study cells, tissues and even small multicellular organisms in conditions that more closely mimic nature, enabling longer viewing times and less adulterated results. Their technique—known as light sheet microscopy—has produced fascinating breakthroughs, allowing them to see live samples longer and clearer than ever before.

The researchers began by turning microscopy on its side—literally. In traditional fluorescence microscopy the whole sample is usually lit by the laser from above. A cameralike detector focuses on successive planes through the sample, snapping pictures from top to bottom that can be stacked into a 3-D picture. But throughout this process the full force of the laser blasts even cells whose light is not in focus for the detector. What the EMBL team realized was that if they swung the laser 90 degrees, so it shined through the sample from the side instead of from above, they could illuminate just the single slice on which the camera was focusing. None of the other cells were hit.

Suddenly, scientists could make movies that lasted for days. The cells under their microscopes, hit with only one five-thousandth of the energy used in traditional fluorescence microscopy, kept on dividing. The EMBL team took a record 24-hour-long movie of a developing zebra fish embryo. When one of the members presented the data at a conference, he was received like a rock star.

But this was only the beginning—the new setup enabled Stelzer's team to use a much faster camera, recording about 60 million pixels per second. And the benefits multiplied: Because the camera was so sensitive, it could absorb more light in each picture, providing even more data.

For scientists it was like going from a choppy, two-minute YouTube clip to a feature film on a flat-screen TV.

The latest breakthrough, published July 30 in Nature Methods, hooks that TV up with high-def cable, if you will. (Scientfic American is part of Nature Publishing Group.) Many biological specimens, like the fruit fly embryo, are so opaque that they scatter large numbers of photons, filling pictures with static. But now, using a technique called structured illumination, the EMBL team has managed to subtract out this interference, making light sheet microscopy even more powerful.

Philipp Keller, who is the lead author of the paper and has joined Howard Hughes Medical Institute since completing the study, compares the process to reconstructing a landscape seen through a blizzard. "All these snowflakes are passing through your field of vision, so parts of the landscape are obscured by them. But if you record multiple images of that landscape, then the snowflakes will have moved on and you can see what was behind them. By recording enough images, you can combine them into a complete reconstruction of the landscape."

For biologists who study development light sheet microscopy, especially using structured illumination, is a godsend.

Kees Weijer of the University of Dundee in Scotland, who studies the migration of cells in chick embryos and slime mold, has been waiting for a light sheet microscope since the EMBL group first publicized its advances. After years of working with other techniques he is thrilled to finally have a scope built for him by Stelzer's lab.

"Light sheet microscopy is a big improvement," he says. "The image quality is just so much better."

"It's really, really groundbreaking," says Michael Davidson of the Florida State University, whose program developed the microscopy Web sites Molecular Expressions, Nikon's MicroscopyU and others. But the addition of structured illumination, Davidson thinks, will be the real game-changer. "That thing is going to take off like a rocket over the next three or four years. It's the first step in what's going to be a long road."

While they wait for a commercial version to come on the market—the technology has been licensed to Germany-based Carl Zeiss optics company—biologists must build each light sheet microscope themselves. At a conference in Dublin on September 2 and 3, researchers interested in the technique plan to meet to swap tips, share ideas and discuss the future.

Keller emphasizes that despite the EMBL group's successes, light sheet microscopy can be used for much more than imaging embryos. With longer observation times, faster images and clearer pictures, he says, great advances in many fields of biology are possible. "It's not a technique that's been developed for a specific purpose. It can go far beyond that," he says. "The discussion was about how to improve microscopy, and this was the result."